Typical procedures for analyzing biological materials, such as nucleic acid, involve a variety of operations starting from raw material. These operations may include various degrees of cell purification, lysis, amplification or purification, and analysis of the resulting amplification or purification product.
As an example, in DNA-based blood tests the samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells. Then, the remaining white blood cells are lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed.
Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample.
The procedures are similar when RNA is to be analyzed, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA.
Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof. In an analysis by hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide probes that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the probes, stable bonds will be formed between them and the hybridized detectors can be read by a wide variety of means, including optical, electrical, magnetic, mechanical or thermal means.
Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody or by an enzymatic reaction of some sort. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways.
It is also known that the most sensitive method to determine the amount of a specific DNA in a sample is the so-called real-time PCR, where the amount of product is measured during ongoing amplification.
During the past few years a number of technologies for simultaneous amplification and detection have been developed. In the simplest assay, the PCR product is detected through the binding of double-strand DNA specific dyes. These kind of dyes have no fluorescence of their own, but become intensively fluorescent when they bind to nucleic acids. However, most are not sequence specific, but will bind to any double-stranded nucleic acid, including the commonly formed, but diagnostically irrelevant, primer-dimer.
A number of oligonucleotide-dye conjugates have been developed that bind via the oligomer to internal DNA sequences and thus allow sequence specific detection. These labels are useful for real-time monitoring of multiplex amplification.
Recently, a new probe for sequence specific detection of target DNA in solution has been proposed (Svanvik N., et al., Detection of PCR Products in Real Time Using Light-up Probes, Analytical Biochemistry 287, 179-182 (2000)). The probe is a peptide nucleic acid to which an asymmetric dye is tethered. Upon sequence specific probe hybridization, the dye also binds to the target DNA, which results in a large increase in fluorescence.
The discussion herein has been simplified by focusing on nucleic acid analysis, in particular DNA amplification, as an example of a biological molecule that can be analyzed using the devices of the invention. However, as described above, the invention can be used for real time monitoring of any chemical or biological test.
Recently, monolithic integrated devices of semiconductor material have been proposed, able to process small fluid quantities with a controlled reaction, and at a low cost (see publications EP161985, EP123739, EP193214, US20030057199, applications EP 03103421.8 and EP 03103422.6, both filed on Sep. 17, 2003, all in the name of the present Applicant).
These devices comprise a semiconductor material body accommodating buried channels that are connected, via input and output trenches, to input and output reservoirs, respectively, to which the fluid to be processed is supplied, and from which the fluid is collected at the end of the reaction. Above the buried channels, heating elements and thermal sensors are provided to control the thermal conditions of the reaction. In one embodiment, the output reservoir also contains detection electrodes that are provided for examining the reacted fluid.
An ever-increasing market demand exists for integrated semiconductor chemical microreactors designed to easily allow real-time monitoring of the reaction occurring within the device.
The aim of the present invention is therefore to provide an integrated semiconductor chemical microreactor for real-time amplification monitoring which meets such a market demand.